The ability to detect differences in DNA sequence (i.e. mutations) is central to the diagnosis of genetic diseases and to the identification of clinically significant variants of disease-causing microorganisms. One method for the molecular analysis of genetic variation involves the detection of restriction fragment length polymorphisms (RFLPs) using the Southern blotting technique (Southern, E. M., J. Mol. Biol., 98:503-517, 1975; Kan et al., Nature, 313:369-374, 1978; Wyman et al. Proc. Natl. Acad. Sci. USA, 77:6754-6758, 1980). Since this approach is relatively cumbersome, new methods have been developed, some of which are based on the polymerase chain reaction (PCR). These include: RFLP analysis using PCR (Chehab et al., Nature, 329:293-294, 1987; Rommens et al., Am. J. Hum. Genet., 46:395-396, 1990), the creation of artificial RFLPs using primer-specified restriction-site modification (Haliassos et al., Nucleic Acids Research, 17:3606, 1989), and hybridization to allele-specific oligonucleotides (ASOs) (Saiki et al., Nature, 324:163-166 (1986).
These methods are limited in their applicability to complex mutational analysis. For example, in cystic fibrosis, a recessive disorder affecting 1 in 2000-2500 live births in the United States, more than 225 presumed disease-causing mutations have been identified. Furthermore, multiple mutations may be present in a single affected individual, and may be spaced within a few base pairs of each other. These phenomena present unique difficulties in designing clinical screening methods that can accommodate large numbers of sample DNAs.
In U.S. patent application Ser. No. 07/957,205, abandoned and in Shuber et al., Human Molecular Genetics, 2:153-158, 1993, the present inventors disclose a method that allows the simultaneous hybridization of multiple oligonucleotide probes to a single target DNA sample. By including in the hybridization reaction an agent that eliminates the disparities in melting temperatures of hybrids formed between synthetic oligonucleotides and target DNA, it is possible in a single test to screen a DNA sample for the presence of different mutations. Typically, more than 50 ASOs can be pooled and hybridized to target DNA; in a second step, ASOs from a pool giving a positive result are individually hybridized to the same DNA.
This methodology is, however, limited by the necessity of performing subsequent multiple individual hybridizations to identify the relevant ASO from the pool. Thus, there is a need in the art for relatively low cost methods that allow the efficient screening of large numbers of DNA samples for genetic variation and the rapid identification of the variant sequence.